Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy

Abstract

Tauopathy is a type of dementia defined by the accumulation of filamentous tau inclusions in neural cells. Most types of dementia in the elderly, including Alzheimer's disease, are tauopathies. Although it is believed that tau protein abnormalities and/or the loss of its functions results in neurodegeneration and dementia, the mechanism of tauopathy remains obscure. Loss of microtubules and/or tubulin is a known consequence of tau accumulating in neurons in Alzheimer's disease. In other words, there is an excess level of tau relative to tubulin in tauopathy neurons. To test whether this imbalance of tau and tubulin expression results in the neurotoxicity of tau, we developed several transgenic C. elegans lines that express human tau at various levels in pan-neurons. These worms showed behavioral abnormalities in a tau expression-dependent manner. The knockdown of a tubulin-specific chaperon, or a subset of tubulin, led to enhanced tau toxicity even in low-expressing tau-transgenic worms that showed no abnormal behaviors. In addition, the suppression of tau expression in tubulin knockdown worms rescued neuronal dysfunction. Thus, not only the overexpression of tau but also a reduction in tubulin can trigger the neurotoxicity of tau. Tau expressed in worms was also highly phosphorylated and largely bound to tubulin dimers rather than microtubules. Relative amount of tubulin-unbound tau was increased in high-expressing tau-transgenic worms showing tau toxicity. We further demonstrated that tau aggregation was inhibited by co-incubation of purified tubulin in vitro, meaning sufficient amounts of tubulin can protect against the formation of tau inclusions. These results suggest that the expression ratio of tau to tubulin may be a determinant of the tauopathy cascade.

Keywords: Alzheimer’s disease; C. elegans; microtubule; neurodegeneration; tau; tauopathy; tubulin.

FIGURE 1

Tau level-dependent neuronal dysfunction in WT4R-tau-Tg worms. Mock-Tg (a), WT4R(L)-Tg (b), WT4R(H)-Tg (c), WT4R(ExH1)-Tg (d), and WT4R(ExH2)-Tg (e) worms were grown on NGM plates and subjected to (A) Western blotting of tau (pool-2) and tubulin (DM1A) in the total lysate. Expression levels were quantified and shown against WT4R(L)-Tg (Lower panel). Average of four independent experiments are shown (means ± SEM, n = 4). Statistical significance was analyzed by Tukey’s post hoc test (^∗∗p < 0.01, ^∗∗∗p < 0.001, vs. WT4R(L)-Tg). (B) The behavioral analysis of uncoordinated movement in each worm line was analyzed. Upper panel shows the population of healthy (open), weak Unc (hatched), and severe Unc (closed) worms (n = 60) in each line. Lower panel shows that the severity of Unc as described in Materials and Methods section. Average scores of three independent experiments are shown (means ± SEM, n = 3). Statistical significance was analyzed by Tukey’s post hoc test (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, vs. Mock-Tg).

FIGURE 2

Tau expression enhances uncoordinated movement induced by tbce-1 gene knock down. (A) WT4R(L)-Tg/rrf-3 (a–c) and Mock-Tg/rrf-3 worms (d) were grown on L4440::T (a; control) or L4440:: tbce-1 (b–d; tbce-1) RNAi plates from embryo to 3 days after hatch. Rrf-3 background worms grown on tbce-1 RNAi plates showed Unc (b) or a frequent burst phenotype (c). (B) The severity of Unc was assessed as described in Materials and Methods section. Average scores of three independent experiments are shown (means ± SEM, n = 3). WT4R(L)-Tg/rrf-3 worms showed a significantly higher sensitivity to tubulin chaperon knockdown. (C) The levels of tbce-1 mRNA were quantified by quantitative real time RT-PCR (means ± SEM, n = 3). tbce-1 mRNA is suppressed to less than 25% compared to controls, regardless of tau expression. (D) The levels of tau and α-tubulin were analyzed by Western blotting. The percent expression of control worms is shown (means ± SEM, n = 3). Statistical significance was analyzed by Tukey’s post hoc test (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001) or Student’s t-test (^∗p < 0.05).

FIGURE 3

Tau expression in mechanosensory neurons exacerbates touch sense abnormality induced by tubulin knockdown. (A) WT4R(L)-Tg/rrf-3 (a–c) and Mock-Tg/rrf-3 (d–f) worms were subjected to immunolabeling using 6-11B1 (anti-mec-12; a and d), and pool-2 (anti-tau; b and e). Merged views are also shown (c and f). Human tau was expressed in as pan-neuronal pattern (b and e) including PLMs (arrows in a–c) in lumber ganglia and ALMs (arrowheads in d–f) in the abdomen. Scale bars = 30 μm. (B) Mock-Tg/rrf-3 (open) and WT4R(L)-Tg/rrf-3 (solid) worms were grown on L4440::T (control) or L4440::mec-12 RNAi plates from embryo to 4 days after hatch. Touch sensitivity was analyzed as described in the Materials and Methods section (means ± SEM, n = 60). (C) The expression of mec-12 mRNA in the indicated worms was quantified by quantitative real time RT-PCR (means ± SEM, n = 3). Statistical significance was analyzed by Tukey’s post hoc test (^∗p < 0.05, ^∗∗∗p < 0.001).

FIGURE 4

Tau reduction rescues the neuronal dysfunction induced by tubulin knockdown. Mock-Tg/rrf-3 and WT4R(L)-Tg/rrf-3 worms were grown on L4440::T, L4440::mec-12 + L4440::T, or L4440::mec-12 + L4440::tau RNAi plates from embryo to 4 days after hatch. (A) Expression levels of MEC-12, tau and GAPDH were quantified by Western blotting. Lower panel showed the relative expression of Mec-12 and tau against naive worms (means ± SEM, n = 4). (B) The average number of responses per 5 trials of anterior touch are shown (means ± SEM, n = 80). The touch-sense abnormality of WT4R(L)-Tg/rrf-3 induced by mec-12 RNAi was significantly reduced by co-administering tau RNAi. Statistical significance was analyzed by Tukey’s post hoc test (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

FIGURE 5

Human-tau in punc-119::tau worms are not bound to MTs. (A) Flowchart of fractionation of MT and free tubulin fractions from worms are shown. (B) Western blotting of free tubulin (Free) and MT fractions from indicated worms using anti-tau (HT7) and anti-α-tubulin (DM1A) antibodies. (C) The amounts of tau and α-tubulin in Free and MT fractions were quantified (means ± SEM, n = 5). Statistical significance was analyzed by Tukey’s post hoc test (^∗∗∗p < 0.001).

FIGURE 6

In vitro dephosphorylation restores the MT-binding activity of human-tau in tau-Tg worms. (A) l-phosphatase dephosphorylation of tau in worms was assessed by Western blotting using the indicated antibodies. (B) Purified tau from WT4R(H)-Tg worms were remixed with recombinant human 0N4R tau isoform (h-tau, positive control) and MTs from naive worms (total). MT-unbound and MT-bound fractions were prepared from this mixture as described in the Materials and Methods section. Bands corresponding to phosphorylated tau (P-tau, open arrowheads), dephosphorylated tau (DeP-tau, closed arrowheads), and recombinant tau (h-tau, arrows) are indicated. Note that a portion of dephosphorylated tau, but not phosphorylated tau, was recovered in the MT-bound fraction like recombinant tau.

FIGURE 7

MT-unbound tau in tau-Tg worms interacts with α/β-tubulin dimers in the soluble fraction. MT-unbound soluble fractions were prepared from Mock-Tg (a), WT4R(L)-Tg (b), and WT4R(H)-Tg (c) worms and subjected to the immunoprecipitation using anti-tau IgG (H-150). (A) Expression of tau, α-tubulin, and α-actin in the total lysate (input) are shown. (B) Immunoprecipitated proteins were analyzed using anti-tau (HT7), anti-α-tubulin (DM1A), and anti-β-tubulin (KMX-1) antibodies. (C) Relative amount of tau and α/β-tubulin in the co-immunoprecipitaed fraction from tau-Tg worms were quantified (mean ± SEM, n = 6). (D) Quantitative analysis of each protein indicates that the nearly equal amount of α- and β-tubulin were co-immunoprecipitated with tau. Std. indicates the purified recombinant tau (for HT7) or porcine tubulins (for DM1A and KMX-1). Statistical significance was analyzed by Tukey’s post hoc test (^∗∗p < 0.01).

FIGURE 8

TMAO improves behavioral phenotypes of tau-expressing worms. (A) Mock-Tg or WT4R(H)-Tg worms were treated with TMAO for 4 days and evaluated for Unc. The data indicate the populations of severely affected worms (means ± SEM, n = 5). (B) Total lysates from TMAO treated worms were analyzed by Western blotting using antibodies indicated. (C) The amounts of tau and α-tubulin were quantified (means ± SEM, n = 7). Statistical significance was analyzed by Tukey’s post hoc test (^∗p < 0.05).

FIGURE 9

Tubulin inhibits heparin-induced aggregation of tau. Recombinant tau (a–d) and tubulin (0.2 mg/ml; c, 1.0 mg/ml; d) were incubated without (closed: a) or with (open: b–d) heparin at indicated periods. (A) Time courses of Th-T fluorescence change are shown (means ± SEM, n = 4). The statistical significance compared to controls (+heparin, 0 mg/mL tubulin) was analyzed by Tukey’s post hoc test (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). (B) After 72 hincubation, the proteins were subjected to the Sarkosyl-solubility assay as described in the Materials and Methods section. Gel images of CBB staining of total, Sarkosyl-soluble, and Sarkosyl-insoluble fractions are shown. Arrows and arrowheads indicate tau and tubulin, respectively. Note that the Sarkosyl-insoluble tau was reduced in the presence of tubulin (the arrow in lowest panel). (C) The amounts of Sarkosyl-insoluble tau were quantified (means ± SEM, n = 4). Statistical significance was analyzed by Tukey’s post hoc test (^∗p < 0.05, ^∗∗∗p < 0.001).